SU1327511A1 - Method of producing collagen peptides containing section of binding with fibronectin - Google Patents

Abstract

The invention relates to peptides, in particular, ps lumen of collagen peptides, co ;; ep and 1, their binding site to fibrobot 1, which is used in biochemistry. The goal is to simplify the process and turn out the target products. Brom cyan is obtained by fragmentation of collagen followed by chromatography under certain conditions. Chromatography is carried out on sepharose with an immobolized gelatin-binding domain by fibronectin, equilibrated with 0.04-0.06 M tris-HCl buffer solution with pH 7. 4–7.6, containing 0.1–0.15 M sodium chloride at a flow rate of 150–250 ml / h and 37–40 ° C. Then the whole is washed with 1–1.5 M urea in a weighting buffer. , and elution of collagen peptides containing the fibronectin binding site is carried out with 4-8 M pa in urea in the above buffer solution. The method allows to reduce the number of stages from 5 to 2, the process time is three times and increase the yield from 5 to 25%. SB ts9 sp

Description

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The invention relates to an improved method for the preparation of collagen peptides containing a fibronectin binding site, biologically active compounds that are used in biochemistry.

The purpose of the invention is to simplify the process of collagen peptides containing the fibronectin binding site to increase the yield of the target product.

Example I. Preparation of d -CB 7 Bromine Peptide Collagen.

A. Brom cyan - fragmentation of the whole collagen molecule. A solution of 180 mg of collagen from rats' skin in 8 ml of 70% formic acid is saturated with N., 200 mg of cyan bromide are added and the mixture is incubated for 5 hours at. The reaction with a MoM% is desalted with an iicL column with biogel P-2 (V 100 ml), equilibrated with 0.1 M acetic acid. Current speed 45 MP / h. The fraction leaving the exclusive volume of the column is lyophilized and 165 mg of a mixture of collagen bromine peptides are obtained,

B. Isolation of 1-CB7 peptide from a mixture of collagen bromide peptides. The resulting mixture of bromine cyanic peptides of collagen (165 mg) is dissolved in 20 ml of 0.05 M Tris-HC pH 7.4, with 0.15 M sodium chloride when heated to and the solution is stirred at 20 ° C for 1 h with 15 ml Sepharose 4B containing immobilized gelatin-binding domain of fibronectin (mol 45,000) (the content of the domain in the adsorbent is 1-2 mg / ml of the packaged volume of the gel). Then adsorbed is packaged in a column, washed. equilibration buffer solution (50 ml), then with the same solution containing 1 M urea (50 ml). The flow rate is 200 ml / h. d 1-CB7 peptide is eluted with a balancing buffer solution containing 4 M urea. The fraction containing about 1-СБ7 peptide is desalted on a column with P-2 biogel equilibrated with 0.1 M acetic acid and lyophilized. OUTPUT / 1-CB peptide 7.5 mg -. (25%). The product was electrophoretic homogeneous upon electrophoresis in a polyacrylamide gel in the presence of sodium dodecyl sulfate (mol. Weight about 25,000).

At a flow rate of 150 b / h, a temperature of 37 ° C, o I-GB7 peptide is obtained with a yield of 27%.

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SECTION 2. Obtaining nascent-Hbix collagen peptides containing the binding site for fibropectinone.

A solution of a mixture of nascent P | „-peptides obtained in a cell-free system of protein biosynthesis and labeled with H-proline (total radioactivity 1.5-10 disintegrations / min) in 3 ml of 0.05 M tris-HCl, pH 7.6 , with 0.15 M sodium chloride, incubated for 1 hour with a 3 ml gelatin-binding fibronectin domain immobilized on separoea 4B. The adsorbent is packed in a column, washed with 40 ml of a balancing buffer solution until no radioactivity is present in the eluates. The flow rate is 200 ml / h. The column is then washed with 1 M urea in a equilibrating buffer solution (15 ml) and nascent peptides, collagen containing a fibronectin binding site, are eluted with B and Urea in a equilibrating buffer solution. The total radioactivity of the obtained fraction (6 ml) is 5-10 disintegrations / min. After dialysis of this fraction against 0.05 M Tris-HC1 buffer solution, pH 7.6, containing 1 M sodium chloride, the target nascent peptides are analyzed by a collagenase reaction.

Similar results were obtained when conducting chromatography of a mixture of nascent peptides at a flow rate of 250 ml / H.

10 µl of collagenase solution (1-5 µg of protein), free from admixture of other proteases, was added to O, I ml of the n-peptide labeled N-proline (MO disintegration / min) obtained after chromatography. Control samples do not contain collagenase. Samples incubated for 90 min at 37 ° C, after which the peptides are precipitated with an equal volume of 10% trichloroacetic acid with 0.5% tannin. The precipitates are collected on filters (Millipor), dried and the radioactivity is determined in a scintillation counter. The cleavage of P „- peptides is calculated by the formula

.p i-i

r, / .- iuu -,

where 0 „- radioactivity experienced

samples;

K - radioactivity of control samples.

According to the data obtained, the initial mixture of nascent P? -Peptides was cleaved by collagenase by 15%, while the Nazitite peptides eluted with a () of the finite adsorbent 8 M. from the column, the drug consists of 90% of the collagen peptides containing the fibronectin binding site.

PRI me R 3. A. Obtaining a Fibronectin Desirable Binding Domain.

300 mg of lyophilized fibronectin from a human blood sample are suspended in 60 ml of a 0.025 M Tris-HC buffered solution containing 0.5 mmol of EDTA and 50 mmol of NaCl (pH 7.0), I mg O is added (-chymotrypsin and the mixture is incubated with stirring 1 hour at 37 ° C. Then 3 mg of trypsin inhibitor from soybeans and 1 mmol of phenylmethylsulfonyl fluoride are added to the mixture after 10 minutes, centrifuged at 10,000 rpm for 10 minutes, supernatant applied to a column (60 ml) with gelatin (Sepharose) equilibrated with 0.025 M Tris-HC1 buffer solution with 0.5 mmol EDTA and 50 mmol NaCl (pH 7.0), column rinse with the same buffer solution until no absorption is taken at 280 -IM in the eluates and the fibronectin gelatin-binding domain is eluted by adding uM into the buffer solution A. The corresponding fractions (L-60 ml) are dialyzed against 2L O, M acetic acid. After lyophilization, the gelatin-binding domain is 25 mg, the molar mass is about 45000. During the electrophoresis in I2.5% polyacrylamide hepatitis in the presence of sodium doradyl sulfonate, the product was homogeneous.

B. Obtaining an adsorbent fibronectin domain (mol. Weight 43,000) - Sepharose 4B.

The lyophilized gelatin-binding domain of fibronectin obtained in item A (25 mg) is dissolved in 15 ml of 0.1 M bicarbonate buffer solution, pH 8.4, containing 0.15 M NaCl and mixed with 15 ml of BrCN-activated Sepharose 4B (activated by the standard procedure using 2 g of BrCN per 10 ml of packed volume of sepharose) for 16 hours at 4 s. The sorbent is washed with 50 ml of the same buffer solution.

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50 ml of boile number 1 and stirred for 2 hours at 20 ° C with 15 ml of a 1 M solution of ethanolamine (pH 9 with 6N HCl). The syrbent is separated and the trid1 1 is washed with 50 ml of O each, 1 M sodium acetate buffer solution with 1 M NaCl, pH 4.5 and 0.1 M and sodium borate buffer solution with 1 M NaCl, pH 9. After washing with water, the sorbent is stored in as a suspension in water in the presence of 0.02% sodium azide. The amount of attached domain is determined by the differential method by the difference in absorbance at. 280 of the original ligand solution and wash water after addition. The sorbent contained 1.5 mg domain per I ml of the packaged volume of the gel.

Such parameters of the chromatographic process as the geometry of the column or the conditions for the subsequent desalting of the corresponding fractions do not affect the final results. The desalting of the fractions was carried out on columns with biogel P-2, equilibrated with 0.1 M acetic acid, at a flow rate of 30-50 ml / h and a volume ratio of the injected sample to the total column volume of 1: 5. The same results were obtained when using C-25 Sephadex for desalting. As regards the flow rate in affinity chromatography, the chromatography of denatured collagen and collagen peptides is usually carried out at. temperature up to 40 ° С and at high current velocity due to the tendency of these biopolymers to renaturation and gelation. Therefore, it is advisable to maintain the current rate in the described process in the range of 150-250 ml / h. The choice of a balancing buffer solution (.0.05 M Tris-HC j pH 7.4-7.6 with 0.1-0.15 NaCl) is caused by the need to create on the column optimal conditions for the interaction of the immobilized domain with peptides containing the binding site with fibronectin. When using 0.04-0.06 M buffers, the results of the separation are almost identical, as in the case of using 0, i-0.15 M NaCl concentrations. The use of NaCl concentrations less than 0.1 M leads to less pure peptides containing the binding site with fibronectin, and more than 0.15 M to the elution of part of these peptides when washing the column with equilibrating buffer solution and

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1 N urea in the same buffer, which reduces the yield of the target product.

The stage of washing the column with 1 M urea is intended to remove from the column of non-decipically adsorbed peptides that does not contain a fibronectin binding site. Urea solutions in concentrations were used for the purpose.} - 1.5 M. At a concentration of urea below 1 M, the non-specific peptide is not completely removed, the target product is not completely contaminated at a concentration greater than 1.5 M, partial elution of collagen peptides containing the area binding to fibronectin, which reduces the yield of target products. The same applies to the elution conditions, since, according to biochemical data, in U, urea effectively divides the biospecific interaction of fibronectin and collagen (gelatin). When using a urea concentration less than in the described process, it is possible to wash out peptides that are not specifically associated with the sorbent due to electrostatic or hydrophobic interactions.

PRI and MER 4. A solution of bromine cyanide peptides on the 1-chain of collagen (100 mg in 12 ml of 0.06 M tris-HCl, pH 7.5 with 0.1 M sodium chloride is stirred at 10 h ml of CL-4B Sepharium containing immobilized. A new gelatin-binding domain of fibronectine (1 mg / ml of packaged volume), the sorbent is packed in a column, washed with a balancing buffer solution (50 ml), then with the same solution containing 1.5 M urea (30 ml). The flow rate is 150 ml / h. Targeted o (1- CB7 peptide is eluted in equilibrium

Compiled by V. Volkov Editor T. Gerasimov Tehred I. Veres

Order 3392

Circulation 348 Subscription

VNIIPI USSR State Committee

for inventions and discoveries 113035, Moscow, Zh-35, PayllIc a nab. d. 4/5

Production and polygraphic enterprise, Uzhgorod ;, st. 11p1N ktp,

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Sivusa buffer solution containing SCHU1M 6M urea. The corresponding fraction is desalted on a column with biogel R-2, balanced with O, M acetic acid, lyophilized. Output about (1-CB7 peptide 4.4 mg (24.5%) i) The product is identical with the 1-CB7 peptide obtained in Example 1.

The described method of obtaining us-. Centrifugal peptides containing the fibronectin binding site reduce the number of stages from 5 to 2, the process time is reduced three times, and the yield is increased five times (from 5% to 25%).

Claims (1)

Invention Formula The method of obtaining collagen peptides containing a binding site either with fibronectin, including collagen bromo-antifragmentation, and subsequent chromatography of a mixture of bromine cyan peptides using buffer solutions as an eluent, characterized in that, in order to simplify the process and yield , chromatography was carried out on Sepharose with an immobilized fibronectin gelatinous domain, yrav with a total of 0.04-0.06 M tris-HC1 buffer solution with a pH of 7.4-7.6, containing 0.10-0.15 M chloride sodium at a flow rate of 150-250 ml / h temperature of 37-40 ° C, followed by washing the column with 1-1.5 M urea in the equilibration buffer solution, elution of collagen peptides containing the fibronectin binding site is carried out with 4-8 M solutions of urea in the same buffer solution. Proofreader A.T. sko